PROJECT SUMMARY/ABSTRACT Pathologic hemorrhage is a major contributor to morbidity and mortality for both the general population as well as individuals with bleeding disorders. While major improvements to human health have been made with blood products and human derived or recombinant coagulation factors, these have limited shelf life and storage conditions, and require intravenous infusion. There are exceedingly few potent and stable conventional pharmaceutical agents for treatment of hemorrhage, and there is a strong need for the identification of new therapeutics and targets. This project will leverage highly innovative technologies, including genome editing nucleases, next generation sequencing, and the zebrafish model to delve into the evolutionary conservation of hemostasis, and use this information to conduct an in vivo high throughput analysis of small molecules and genetic targets for treatment of a hemorrhagic phenotype. In preliminary studies, we have used robust genome editing nucleases (TALENS and CRISPR/Cas) to generate mutations in procoagulant factors. These mutants display bleeding phenotypes and lethality, correlating with data from mammals. In Specific Aim 1, we will use genome editing nuclease to evaluate combinatorial actions of multiple coagulation factors for suppression of a model of hemorrhage. In Specific Aim 2 we will perform a high throughput small molecule screen to detect lead compounds for treatment of hemorrhage, and a sensitized ENU mutagenesis suppressor screen to identify potential modifier genes as new therapeutic targets. These studies will discern interactions amongst candidate modifiers as well as perform an unbiased screen for novel therapeutic drugs and target genes. This will lead to potential innovative agents and therapeutic classes for treatment of hemorrhage that could benefit the general population, as well as patients with bleeding disorders.